SUMMARY Single cell analysis is emerging as an important field of research as technologies improve in sensitivity and with increased throughput to allow measurement and understanding of cell heterogeneity in complex biological systems. The goal of this project is to create the ability to identify and quantify biomolecules inside a single living cell with minimal perturbation and over long periods of time using electrochemical techniques. Intracellular electrochemistry can be accomplished using a novel multifunctional carbon nanotube-based sensor consisting of both working and reference electrodes at a single cell-penetrating tip, through which fluids can be simultaneously injected. In this project, the multifunctional nanoprobe is constructed, capable of injecting fluids in order to conduct self-contained electrochemical measurements inside confined aqueous microenvironments. The nanoprobe is then utilized to inject a reaction-enabling substrate into the cell in order to selectively quantify senescence-associated beta-galactosidase, a cytosolic enzyme of critical importance in many chronic diseases. We hypothesize that CNT-based nanoprobes will provide the ability to selectively quantify cell senescence in a heterogeneous population of living cells in real-time; capabilities not currently available with state-of-the-art technologies. We plan to pursue the following two Specific Aims: (1) construct a multifunctional nanoprobe capable of injecting fluids in order to conduct self-contained electrochemical measurements inside confined aqueous microenvironments. Here, we will manufacture the CNT-based probe and develop the techniques needed to utilize the probe for intracellular electrochemistry; (2) utilize the nanoprobe to inject p-aminophenyl ?-D-galactopyranoside in order to selectively quantify senescence- associated ?-galactosidase. Here, we will use the CNT probe to electrochemically measure senescence- relevant molecules inside individual living cells in vitro and evaluate the measurement capabilities of the probe with standard detection assays. The outcome of this proposal will provide a first-in-class CNT-based tool that (a) establishes a new single cell analytical technique (intracellular electrochemistry) and (b) combines it with traditional techniques (intracellular injection and fluorescence microscopy) to form an entirely new minimally invasive analytical technique for gathering quantitative data from single living cells with high temporal resolution and over long periods of time. Moreover, the new nano-biosensing tool and technique can be efficiently disseminated to the larger scientific community to assist researchers in studying and elucidating fundamentals in cell biology. Real-time intracellular electrochemistry measurements will have great translational potential in the development of diagnostic and therapeutic approaches for several diseases, including chronic obstructive pulmonary disease (COPD), lung cancer and cardiovascular disease, all of which are major public health concerns.